All proteins for crystallizations and in vitro experiments were expressed in E. coli BL21 (DE3). After cultivating bacteria at 37°C until OD600 reached 0.8 to 1.2, overnight culturing with 100 μM IPTG was performed at 16°C. After centrifugation, the bacteria were resuspended to PBS with 5 mM EDTA and lysed by sonication for 10 min. After centrifugation, the supernatants were incubated with affinity resin column: GST accept resin (Nacalai Tesque) for GST-fused proteins and Amylose Resin High Flow resin for MBP-fused proteins (New England Biolabs). After washing the resin with PBS three times, the proteins were eluted with glutathione buffer (10 mM glutathione and 50 mM Tris-HCl pH 8.0) for GST-fused proteins or maltose buffer (10 mM maltose, 20 mM Tris-HCl pH 8.0, 200 mM sodium chloride) for MBP-fused proteins. The eluates were then digested by HRV 3C protease at 4°C for overnight to remove the affinity tag. The proteins were further subjected to size exclusion chromatography (SEC) with 20 mM HEPES pH 6.8 and 150 mM sodium chloride by using Superdex 75 26/60 or Superdex 75 10/300 column (GE Healthcare). Synthesized SpHfl1(386-398) peptide (purchased from Bex Co.) was dissolved in water and purified by SEC with 20 mM HEPES pH 6.8 and 150 mM sodium chloride by using Superdex peptide 10/300 column (GE Healthcare).
Superdex peptide 10 300 column
Manufactured by GE Healthcare
Sourced in United States
The Superdex peptide 10/300 column is a size exclusion chromatography column designed for the purification and analysis of peptides and small proteins. It features a porous matrix that separates molecules based on their size and molecular weight. The column has a bed volume of 24 mL and is compatible with a variety of buffer systems, making it a versatile tool for researchers working with peptides and small proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 c5 maleimide, by Thermo Fisher Scientific (2 mentions)
Superdex 75 10 300 column, by GE Healthcare (1 mentions)
Gst accept resin, by Nacalai Tesque (1 mentions)
Superdex 75 26 60, by GE Healthcare (1 mentions)
1260 infinity 2 hplc system, by Agilent Technologies (1 mentions)
Bradykinin fragment 1 7, by Merck Group (1 mentions)
Leu gly, by Merck Group (1 mentions)
Glycine, by Fujifilm (1 mentions)
Aprotinin, by Fujifilm (1 mentions)
Kta fplc system, by GE Healthcare (1 mentions)
Hplc system, by Shimadzu (1 mentions)
12 protocols using superdex peptide 10 300 column
1
Affinity-Based Protein Purification and Characterization
2
Peptide Sequence Identification by LC-MS
3
Peptide Fractionation and LC-MS/MS Analysis
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Dried peptides from both
experiments were resuspended in 100 μL of 0.1% formic acid by
water bath sonication without cleanup. A Superdex Peptide 10/300 column
(GE Healthcare) was equilibrated with 0.1% formic acid. Two separate
isocratic runs were performed at a flow rate of 0.2 mL/min. Early
fractions were collected starting at 10 min after injection (total
run time of 130 min) and were analyzed by LC-MS/MS as described in
the following sections.
experiments were resuspended in 100 μL of 0.1% formic acid by
water bath sonication without cleanup. A Superdex Peptide 10/300 column
(GE Healthcare) was equilibrated with 0.1% formic acid. Two separate
isocratic runs were performed at a flow rate of 0.2 mL/min. Early
fractions were collected starting at 10 min after injection (total
run time of 130 min) and were analyzed by LC-MS/MS as described in
the following sections.
4
Fluorescent Labeling of Cys-OmpX Protein
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Purified Cys-OmpX was covalently labelled with Alexa Fluor 488 dye via maleimide chemistry. A sample containing 200 μM Cys-OmpX in 25 mM Tris-HCl, 6 M Gdn-HCl, pH 7.2, was incubated with 10 mM DTT for 30 min. This sample was subsequently buffer exchanged into 25 mM Tris-HCl, 6 M Gdn-HCl, pH 7.2 (that had been sparged for 15 min with nitrogen gas) using Zeba spin desalting columns (Thermo Fisher Scientific). Alexa Fluor 488 C5 maleimide (Thermo Fisher Scientific) (10 mg/mL dissolved in DMSO) was immediately added to the OmpX sample at a final concentration of 2 mM. The total sample volume was 480 µL. The labelling reaction was kept at 25 °C for 1 h then left overnight at 4 °C. The reaction was then loaded onto a Superdex Peptide 10/300 column (GE Healthcare) equilibrated with 6 M Gdn-HCl, 25 mM Tris-HCl, pH 7.2 to remove the excess free dye. Samples were collected every 1 mL and peak protein fractions tested for dye labelling using a Nanodrop 2000 (Thermo Fisher Scientific). Samples containing labelled OmpX were snap-frozen using liquid nitrogen and stored at −80 °C until required.
5
Fractionation and Analysis of Peptide Mixture
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Dried peptide mixture without cleanup (2 mg) was dissolved in 100 μl of 0.1% formic acid by vortexing and water bath sonication for 1 minute. The resulting 100 μl was put in the sample manager of Agilent Infinity 1260 II HPLC system. Superdex peptide 10/300 column (GE Healthcare) was equilibrated for 2 hours with 0.1% formic acid as solvent. A flow rate of 0.2 ml min−1 was used throughout the isocratic run using 0.1% formic acid. A total of 48 fractions were collected starting at 10 minutes after injection until the end of the run (total run time of 130 minutes). 20 μl of each fraction was analyzed by LC-MS/MS as described in following sections.
6
Peptide Molecular Weight Characterization
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The MWD of the peptides after hydrolysis was characterized using GPC. Hydrolysate samples were dissolved in 5 mg/mL HPLC-eluent, and undissolved particles were removed by centrifugation (5 min at 13,000× g) if required, followed by filtration (0.45 μm cellulose acetate filter). Size exclusion chromatography was performed with a Superdex Peptide 10/300 column (GE Healthcare) at 30 °C, with a flow rate of 0.9 mL/minute using an injection of 20 μL of a 5 mg/mL HPLC-eluent (7.4 g trifluoro acetic acid and 1173 g acetonitrile in 3500 g H2O) solution. The column was calibrated with 10 peptide standards: cytochrome C (M = 12,327), aprotinin (M = 6500), adrenocorticotropic hormone from porcine pituitary gland (M = 4567), insulin A-chain oxidized ammonium salt from bovine pancreas (M = 2532), angiotensinogen 1–14 renin substrate porcine (M = 1759), bradykinin salt (M = 1060), bradykinin fragment 1–7 (M = 757), bradykinin fragment 1–5 (M = 573), Ala-Ala-Ala-Ala-Ala (M = 373), and Gly-Leu (M = 188), all of which were from Sigma Aldrich, St. Louis, MO, USA. The eluate was monitored at 200 nm.
7
Size Exclusion Chromatography of CLE Digest
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The artificial CLE digest was fractionated by size exclusion chromatography (SEC) using Superdex peptide 10/300 column (GE Healthcare, Amersham Place, UK) at 25℃. The mobile phase was 50 mM phosphoric acid and 150 mM sodium chloride and the flow rate was 0.5 mL/min. Aprotinin (molecular weight (MW): 6,512, FUJIFILM Wako Pure Chemical Corporation), angiotensin II (MW: 1,046, FUJIFILM Wako Pure Chemical Corporation), triglycine (MW: 189, Tokyo Chemical Industry, Tokyo, Japan) and glycine (MW:75, FUJIFILM Wako Pure Chemical Corporation) were used as standards. An analytical curve of the molecular weight and the elution time was made and the average molecular weight was calculated.
8
Dansylation and Purification of UDP-MurNAc-pentapeptide
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Synthesis and purification UDP-MurNAc-pentapeptide was achieved by chemoenzymatic recapitulation of the cytoplasmic synthetic pathway in vitro43 (link). Desalted UDP-MurNAc-pentapeptide in sterile water was mixed with an equal volume of acetone and allowed to react overnight with a 42-fold molar ratio of dansyl chloride with stirring. The reaction was quenched with a 10-fold molar excess of Tris-Cl pH 9 to dansyl chloride before rotary evaporation to remove solvents. Dried products were resuspended in 1 ml sterile water and purified by size exclusion chromatography on a Superdex peptide 10/300 column (GE Healthcare) pre-equilibrated with 1.5 CV 0.1 M ammonium bicarbonate. Dansylated UDP-MurNAc-pentapeptide is the first peak to elute in this procedure with a characteristic absorbance at 340 and 280 nm. Fractions containing the required product were freeze dried 4 times and resuspended in a small volume of sterile water. Quantification and purity were confirmed by absorbance and mass spectrometry, respectively.
9
Cys-tOmpA Labeling with Alexa Fluor 488
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Purified Cys-tOmpA was covalently labelled with Alexa Fluor 488 dye via maleimide chemistry. A sample containing 200 μM Cys-tOmpA in 25 mM Tris–HCl, 6 M Gdn–HCl, pH 7.2, was incubated with 10 mM DTT for 30 min. This sample was subsequently buffer exchanged into 25 mM Tris–HCl, 6 M Gdn–HCl, pH 7.2 (that had been sparged for 15 min with nitrogen gas) using Zeba spin desalting columns (Thermo Fisher Scientific). Alexa Fluor 488 C5 maleimide (Thermo Fisher Scientific) (10 mg/mL dissolved in DMSO) was immediately added to the tOmpA sample at a final concentration of 2 mM. The total sample volume was 480 µL. The labelling reaction was kept at 25 °C for 1 h then left overnight at 4 °C. The reaction was then loaded onto a Superdex Peptide 10/300 column (GE Healthcare) equilibrated with 6 M Gdn–HCl, 25 mM Tris–HCl, pH 7.2 to remove the excess free dye. Samples were collected every 1 mL and peak protein fractions tested for dye labelling using a Nanodrop 2000 (Thermo Fisher Scientific). Samples containing labelled tOmpA were snap-frozen using liquid nitrogen and stored at −80 °C until required.
10
Superdex Peptide Fractionation and LC-MS/MS
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The other aliquot was resuspended in 100 μl of 0.1% formic acid and injected into Superdex peptide 10/300 column (GE Healthcare) as described previously with some modifications.36 , 37 The peptides were separated using an isocratic flow of 0.1% formic acid for 130 min and 24 early fractions were collected starting at 10 min after injection. The fractions were dried and resuspended in 0.1% formic acid for LC–MS/MS analysis.
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